U.S. patent application number 16/940431 was filed with the patent office on 2020-11-12 for sterilization process.
This patent application is currently assigned to Admedus Regen Pty Ltd.. The applicant listed for this patent is Admedus Regen Pty Ltd.. Invention is credited to William Morris Leonard Neethling.
Application Number | 20200353126 16/940431 |
Document ID | / |
Family ID | 1000004978309 |
Filed Date | 2020-11-12 |
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United States Patent
Application |
20200353126 |
Kind Code |
A1 |
Neethling; William Morris
Leonard |
November 12, 2020 |
Sterilization Process
Abstract
The present invention relates to a process for sterilizing
implantable biomaterials. In particular, the invention relates to a
process for sterilizing collagen-containing implantable
biomaterials and storage thereafter.
Inventors: |
Neethling; William Morris
Leonard; (Booragoon, AU) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Admedus Regen Pty Ltd. |
Toowong |
|
AU |
|
|
Assignee: |
Admedus Regen Pty Ltd.
Toowong
AU
|
Family ID: |
1000004978309 |
Appl. No.: |
16/940431 |
Filed: |
July 28, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15150025 |
May 9, 2016 |
10758642 |
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16940431 |
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13561787 |
Jul 30, 2012 |
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15150025 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61L 2202/21 20130101;
A61L 27/3604 20130101; A61L 27/24 20130101; A61L 27/3687 20130101;
A61L 2400/02 20130101; A61K 41/10 20200101; A61L 2/0088 20130101;
A61L 2430/20 20130101 |
International
Class: |
A61L 27/24 20060101
A61L027/24; A61L 2/00 20060101 A61L002/00; A61L 27/36 20060101
A61L027/36; A61K 41/10 20060101 A61K041/10 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 10, 2011 |
AU |
2011904681 |
Claims
1. A method for producing a sterilized calcification-resistant
biomaterial, the method comprising: (a) contacting a biomaterial
with a cross-linking solution, wherein the biomaterial is a
collagen-based biomaterial; (b) rinsing the biomaterial with a
rinsing solution; (c) incubating the biomaterial in a sterilization
solution within a storage container, the sterilization solution
comprising 3% to 6% v/v propylene oxide at an incubation
temperature between about 30.degree. C. and 55.degree. C., wherein
the sterilization solution does not include alcohol.
2. The method of claim 1, further comprising: (d) storing the
biomaterial in a storage solution in the same storage container,
where the storage solution has resulted from the conversion in situ
of the sterilization solution in the storage container in the
presence of the biomaterial.
3. The method of claim 1, wherein the cross-linking solution
comprises a glutaraldehyde solution.
4. The method of claim 3, wherein the cross-linking solution
further comprises potassium di-hydrogen phosphate.
5. The method of claim 1, wherein the cross-linking solution
comprises a cross-linking agent selected from the group consisting
of divinyl sulfone, polyethylene glycol divinyl sulfone,
hydroxyethyl methacrylate divinyl sulfone, formaldehyde,
glutaraldehyde, aldehydes, isocyanates, alkyl halides, aryl
halides, imidoesters, N-substituted maleimides, acylating
compounds, carbodiimide, hydroxychloride, N-hydroxysuccinimide.
6. The method of claim 1, wherein the rinsing solution comprises
sodium chloride.
7. The method of claim 1, wherein the storage solution comprises 3%
to 6% v/v propylene glycol.
8. The method of claim 7, wherein the storage solution comprises
between 3.8% and 4.5% v/v propylene glycol.
9. The method of claim 8, wherein the storage solution comprises
4.4% propylene glycol.
10. The method of claim 1, wherein the collagen-based biomaterial
is isolated from an ovine, a bovine, a caprine, an equine, a
porcine, a marsupial or a human.
11. The method of claim 1, wherein the collagen-based biomaterial
comprises cross-linked collagen-based biomaterial comprises
cellular tissue selected from the group consisting of
cardiovascular tissue, heart tissue, heart valve, aortic roots,
aortic wall, aortic leaflets, pericardial tissue, connective
tissue, dura mata, dermal tissue, a vascular tissue, cartilage,
pericardium, ligament, tendon, blood vessels, umbilical tissue,
bone tissue, fasciae, and submucosal tissue and skin.
12. The method of claim 1, wherein the collagen-based biomaterial
further comprises synthetic analogs formed from synthetic polymers,
biological polymers, or both.
13. The method of claim 1, wherein the biomaterial is incubated for
a period of at least 48 hours.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation application of U.S.
application Ser. No. 13/561,787, filed Jul. 30, 2012, which claims
the benefit of Australian Patent Application No. 2011904681, filed
Nov. 10, 2011, the entire contents of the aforementioned
applications are hereby incorporated herein by reference.
FIELD
[0002] The present invention relates to a process for sterilizing
implantable biomaterials. In particular, the invention relates to a
process for sterilizing collagen-containing implantable
biomaterials and storage thereafter.
BACKGROUND
[0003] Implantable biomaterials, especially collagen-based
biomaterials, require sterilization and most often storage before
use. Generally there are two broad classes of implantable
collagen-based biomaterials: (1) natural tissue and (2) chemically
cross-linked tissue. Thus, depending upon the type of
collagen-based biomaterial and whether or not cross-linking has
taken place there is a need for a means of sterilizing the tissue
as well as storing tissue once it has been sterilized.
[0004] Chemical cross-linked collagen-based biomaterials such as
cardiovascular patches, heart valves, matrices and arteries are
usually sterilized after cross-linking and stored in a sterile
solution until implantation. Several sterilization methods for
chemical cross-linked collagen-based biomaterials have been tested
and implemented over the past three to four decades including gamma
irradiation, UV irradiation and a variety of chemical agents.
Although most of these sterilization methods are efficient in
preventing contamination, adverse effects such as structural damage
(cleaving of peptide bonds) and tissue degeneration (reduction in
tensile strength) has made a number of these methods less appealing
for industrial application.
[0005] For example, collagen-based biomaterials cross-linked with
glutaraldehyde can become chemically unstable when exposed to
alcohol-based sterilisation solutions due to the interaction of the
alcohol with residual and unbound glutaraldehyde present in the
tissue. Unstable hemiacetyls are formed when an alcohol reacts with
an aldehyde. These unstable hemiacetyls have the capacity to react
with alcohol to form an acetyl, which can dissociate to form an
aldehyde and an alcohol.
[0006] Thus, at present, the majority of manufacturers of
collagen-based biomaterials prefer the use of
glutaraldehyde-formaldehyde combinations for chemical cross-linking
and non-aldehyde agents for sterilization. One such non-aldehyde
agent is ethylene oxide (oxirane) gas, which has been used to
sterilize mechanical heart valves for many years. Ethylene oxide
gas has also been used to sterilize a variety of medical equipment,
disposable items and mechanical heart valves.
[0007] Once the collagen-based biomaterial has been sterilized it
is generally stored for a period of time before implantation. Mid-
to long-term storage of collagen-based biomaterials requires
adequate protection from contamination in a physiologically, stable
solution. Although most of the commercially available
collagen-based biomaterials are still stored in aldehyde-based
solutions, adverse effects such as calcification and fibrosis are
well known.
[0008] Since the 1970's propylene oxide has been used as a
sterilizing agent (see, for example, Hart & Brown, 1974, Appl
Microbiol, December p. 1069-1070; Brown & Ng, 1975, Appl
Microbiol, September p 483-484). In each case a solution comprising
5% propylene oxide plus 70% isopropyl alcohol or 0.5% chlorhexidine
or 2% Cetrimide was effective in destroying a bacterial spore
suspension. However, while the use of propylene oxide has been
recorded this is usually applied in the presence of alcohol
(ethanol or isopropanol). Thus, the use of an alcohol as an
additive to propylene oxide sterilisation with aldehyde
cross-linked tissues (containing residual aldehydes) could result
in elevated aldehyde levels, which in turn increases the
calcification potential of these tissues and ultimately
bioprosthetic failure.
[0009] Consequently, what is required is an efficient sterilization
process which not only sterilizes chemical cross-linked
collagen-based biomaterials, but also provides a convenient storage
medium for the sterilized biomaterial.
SUMMARY
[0010] The inventors have developed a process that overcomes or at
least alleviates the problems associated with typically used
sterilization and/or storage methods for cross-linked
collagen-based biomaterials.
[0011] Accordingly, in a first aspect the present invention
provides a method for sterilizing a cross-linked collagen-based
biomaterial comprising contacting said cross-linked collagen-based
biomaterial with a sterilization solution comprising between 3% and
6% v/v propylene oxide and incubating said biomaterial between
30.degree. C. and 55.degree. C. for greater than 48 hours; with the
proviso that the sterilization solution does not include
alcohol.
[0012] In some embodiments the incubation temperature is between
30.degree. C., 31.degree. C., 32.degree. C., 33.degree. C.,
34.degree. C., 35.degree. C., 36.degree. C., 37.degree. C.,
38.degree. C., 39.degree. C., 40.degree. C., 41.degree. C.,
42.degree. C., 43.degree. C., 44.degree. C., 45.degree. C.,
46.degree. C., 47.degree. C., 48.degree. C., 49.degree. C.,
50.degree. C., 51.degree. C., 52.degree. C., 53.degree. C.,
54.degree. C. and 55.degree. C. In other embodiments the incubation
temperature is between 30.degree. C. and 31.degree. C., 32.degree.
C., 33.degree. C., 34.degree. C., 35.degree. C., 36.degree. C.,
37.degree. C., 38.degree. C., 39.degree. C., 40.degree. C.,
41.degree. C., 42.degree. C., 43.degree. C., 44.degree. C.,
45.degree. C., 46.degree. C., 47.degree. C., 48.degree. C.,
49.degree. C., 50.degree. C., 51.degree. C., 52.degree. C.,
53.degree. C., 54.degree. C. or 55.degree. C. In other words, all
combinations of temperatures between the range 30.degree. C. and
55.degree. C. are envisaged. In some embodiments the incubation
temperature is preferably between 35.degree. C. and 50.degree. C.,
more preferably between 40.degree. C. and 48.degree. C. In some
embodiments the incubation temperature is about 45.degree. C.
[0013] Once the incubation period has lapsed i.e. more than 48
hours have elapsed it is permissible to allow the temperature to
reduce to room temperature. Indeed, the sterilized cross-linked
collagen-based biomaterial can remain at room temperature for some
time after the initial 48 hours as at this time. Once the
sterilized cross-linked collagen-based biomaterial has been
incubated in the propylene oxide for at least 4 days the propylene
oxide will have been converted to propylene glycol and the
collagen-based biomaterial will be ready to use.
[0014] In some embodiments, the sterilization solution comprises
between 3.8% and 4.5% v/v propylene oxide. In other embodiments,
the sterilization solution comprises about 4% v/v propylene oxide.
In some embodiments, the sterilization solution consists
essentially of between 3% and 6% v/v propylene oxide, more
preferably the sterilization solution consists of between 3% and 6%
v/v propylene oxide. In some embodiments, the sterilization
solution consists essentially of between 3.8% and 4.5% v/v
propylene oxide, more preferably the sterilization solution
consists of between 3.8% and 4.5% v/v propylene oxide. In some
embodiments, the sterilization solution consists essentially of
about 4% v/v propylene oxide, more preferably the sterilization
solution consists of about 4% v/v propylene oxide.
[0015] It will be appreciated that alcohol, especially ethanol
and/or isopropanol is not used in the sterilization solution of the
present invention.
[0016] It is a requirement that the sterilization step is carried
out for greater than 48 hours (2 days); however, as the
sterilization solution can also be used as a storage medium the
sterilization step can be carried out for 2, 3, 4, 5, 6, 7, 8, 9,
10 or more days.
[0017] The cross-linked collagen-based biomaterial can be any
material which comprises, consists essentially of or consists of
collagen. In some embodiments, the collagen-based biomaterial is
isolated directly from an animal. The biomaterial can be isolated
from any animal, whether from the same species as a recipient or
from an animal of a different species to the recipient. Preferably,
the animal is from one of the mammalian orders i.e. Artiodactyla,
Lagomorpha, Rodentia, Perissodactyla, Carnivora and Marsupialia.
More preferably, the animal is selected from the group consisting
of an ovine, a bovine, a caprine, an equine, a porcine, a marsupial
and a human.
[0018] The biomaterial may be any type of cellular tissue.
Preferably, the cellular tissue is selected from the group
consisting cardiovascular tissue, heart tissue, heart valve, aortic
roots, aortic wall, aortic leaflets, pericardial tissue, connective
tissue, dura mater, dermal tissue, a vascular tissue, cartilage,
pericardium, ligament, tendon, blood vessels, umbilical tissue,
bone tissue, fasciae, and submucosal tissue and skin.
[0019] In some embodiments, the biomaterial is and/or comprises
discrete i.e. isolated collagen, rather than a naturally-occurring
collagen-containing tissue. The discrete collagen may be used in
its isolated state or formed into any medical device or article
known in the art.
[0020] In some embodiments, the biomaterial is a cultured tissue, a
prosthesis containing extra-cellular matrix obtained from an
animal, a reconstituted tissue (e.g. collagen matrix), or the
like.
[0021] It will also be appreciated that the biomaterial might
further comprise synthetic analogs formed from synthetic polymers,
biological polymers, or both, including those generally found in
natural tissue matrices. Suitable synthetic polymers include, for
example, polyamides and polysulphones. Biological polymers can be
naturally occurring or produced in vitro by, for example,
fermentation and the like. In a second aspect, the present
invention provides a method for sterilizing a collagen-based
biomaterial comprising:
[0022] (a) providing a collagen-based biomaterial and washing same
with ice-cold 0.9% v/v saline solution and placing said biomaterial
in ice-cold 0.9% v/v saline/Phenyl-methyl-sulfonyl-fluoride
(PMSF);
[0023] (b) contacting said collagen-based biomaterial with 0.625%
v/v glutaraldehyde solution and potassium di-hydrogen phosphate pH
7.4 and incubating same at about 1-5.degree. C. for at least 5 days
to produce a cross-linked collagen-based biomaterial;
[0024] (c) rinsing said cross-linked collagen-based biomaterial in
sterile 0.9% v/v sodium chloride at approximately 10.degree. C. and
then contacting the cross-linked collagen-based biomaterial with a
sterilization solution comprising between 3.8% and 4.5% v/v
propylene oxide and incubating said tissue between 30.degree. C.
and 55.degree. C. for greater than 48 hours; with the proviso that
the sterilization solution does not include alcohol.
[0025] In a third aspect the present invention provides a method
for storing a sterilized, cross-linked collagen-based biomaterial
comprising contacting a cross-linked collagen-based biomaterial
with a solution comprising between 3% and 6% v/v propylene oxide
and incubating said biomaterial between 30.degree. C. and
55.degree. C. for greater than 48 hours and then allowing the
biomaterial to remain in contact with said propylene oxide until
same converts to propylene glycol; with the proviso that the
solution does not include alcohol.
[0026] In a fourth aspect, the present invention provides a
sterilized, cross-linked collagen-based biomaterial produced by a
method according to the first, second or third aspects.
[0027] It will be appreciated that once the sterilized,
cross-linked collagen-based biomaterial has been obtained by the
methods of the present invention it can be included with
implantable biological devices. Accordingly, in a fifth aspect, the
present invention provides an implantable biological device
comprising a sterilized, cross-linked collagen-based biomaterial
according to the fourth aspect.
[0028] In a further aspect of the present invention the
cross-linked collagen-based biomaterial of the present invention is
contained within a kit for repairing a tissue injury. Thus, in a
sixth aspect the present invention provides a kit for repairing a
tissue injury comprising: [0029] (a) a sterile container having a
sterilized, cross-linked collagen-based biomaterial according to
the fourth aspect or a device according to the fifth aspect; and
[0030] (b) instructions for use on an injured subject.
[0031] In a seventh aspect, the present invention provides a
container comprising a sterilized, cross-linked collagen-based
biomaterial and a 3% to 6% v/v propylene glycol solution, wherein
said propylene glycol has resulted from the conversion in situ of a
3% to 6% v/v propylene oxide solution while in the presence of the
biomaterial.
[0032] The collagen-based biomaterial of the present invention may
be cross-linked by any method know in the art of cross-linking
collagen including, but not limited to, the methods disclosed in
Eyre et al., 1984, Annu. Rev. Biochem. 537, 717-748; Eyre, 1982,
In: Symposium on Heritable Disorders of Connective Tissue (Akeson
et al. eds) pp. 43-58, Mosby, St. Louis, Mo.; Davison &
Brennan, 1983, Connect. Tissue Res. 11, 135-151; Robins, 1982,
Methods Biochem. Analysis, 28, 330-379; Reiser, 1991, Proc. Soc.
Exp. Biol. Med. 196, 17-29; all of which are incorporated herein in
their entirety by reference. However, a preferred method of
cross-linking the collagen-based biomaterial of the present
invention comprises: [0033] (a) exposing a collagen-based
biomaterial to an alcohol-containing solution for at least 24
hours; [0034] (b) exposing said biomaterial in step (a) to a
cross-linking agent; and [0035] (c) exposing said biomaterial in
step (b) to an acidic solution; wherein step (b) and (c) are
sequential to step (a).
[0036] The alcohol-containing solution used in step (a) is
preferably a water-based liquid i.e. is an aqueous solution of
greater than about 50% v/v alcohol, and preferably between 60% to
80% alcohol by volume. Either buffered or non-buffered
alcohol-containing solution can be used; however, it is preferable
that a non-buffered alcohol-containing solution is used as it has
been found that buffered alcohol-containing solutions adversely
affect subsequent cross-linking procedures producing a yellowed
biomaterial.
[0037] The preferred method of cross-linking can use any alcohol
known in art in the alcohol-containing solution. Preferably, the
alcohol is a C.sub.1-C.sub.6 lower alcohol in a buffer-free
solution.
[0038] Even more preferably, the alcohol is selected from the group
consisting of methanol, ethanol, cyclohexanol, isopropanol,
propanol, butanol, pentanol, isobutanol, sec-butanol and
t-butanol.
[0039] In some embodiments, the alcohol-containing solution
comprises a mixture of two or more alcohols provided that the
combined volume of the alcohol is greater than 50% v/v. For
example, a mixture of about 70% v/v ethanol and about 10% v/v
isobutanol is effective.
[0040] The biomaterial in step (a) can be exposed to the
alcohol-containing solution for any length of time as long as it is
sufficient to render the biomaterial resistant to in vivo
pathogenic calcification. Preferably, the biomaterial remains in
contact with the alcohol-containing solution for sufficient time to
enable the alcohol to diffuse and permeate into the biomaterial.
More preferably, the biomaterial is exposed to the
alcohol-containing solution for at least 24 hours, even more
preferably at least 36 hours and most preferably, at least 48
hours.
[0041] The biomaterial, after exposure to the alcohol-containing
solution, is removed and exposed to one or more cross-linking
agents. Any form of cross-linking agent known in the art or
combination thereof may be used as long as it is capable of
cross-linking collagen. Accordingly, it will be appreciated that
cross-linking agents, include but are not limited to, divinyl
sulfone (DVS), polyethylene glycol divinyl sulfone (VS-PEG-VS),
hydroxyethyl methacrylate divinyl sulfone (HEMA-DIS-HEMA),
formaldehyde, glutaraldehyde, aldehydes, isocyanates, alkyl and
aryl halides, imidoesters, N-substituted maleimides, acylating
compounds, carbodiimide, hydroxychloride, N-hydroxysuccinimide,
light (e.g., blue light and UV light), pH, temperature, and
combinations thereof. Preferably, the cross-linking agent is a
chemical cross-linking agent selected from the group consisting of
carbodiimide, polyepoxy ethers, divinyl sulfone (DVS), polyaldehyde
and diphenylphosphoryl azide (DPPA).
[0042] In some embodiments, the polyaldehyde is a bi-, tri- or
di-aldehyde. Glutaraldehyde is especially preferred.
[0043] In some embodiments, the cross-linking step (b) is followed
by step (c), with or without an intervening wash step. The acidic
solution used in step (c) contains any acid capable of inactivating
and/or modifying the fixed and/or unfixed cross-linking agent
moieties present in the biomaterial after step (b) to remove or
reduce available calcium binding sites. Alternatively, or in
addition to, the acidic solution used in step (c) contains any acid
capable of further cross-linking the activated carboxyl groups with
the activated amine groups on the collagen to form amide bonds.
Preferably, the acid in the acidic solution comprises an
aminocarboxylic acid. Preferably, the aminocarboxylic acid is an
acid having at least one amino group and at least one carboxylic
acid substituent. More preferably, the aminocarboxylic acid is
selected from the group consisting of L-arginine, L-lysine,
L-histidine, L-glutamate or L-aspartate.
[0044] The step of rinsing the biomaterial is conducted using a
phosphate-free solution of 0.9% v/v saline.
[0045] While it will be appreciated by those skilled in the art
that the temperature at which each of the steps in the preferred
cross-linking method is carried out is not critical, it will be
understood that preferably, the temperature is between 2.degree. C.
and 40.degree. C., more preferably, between 4.degree. C. and
30.degree. C. and most preferably, between 5.degree. C. and
25.degree. C.
[0046] In one embodiment, the alcohol, acidic solution and rinsing
solution are all buffer-free.
BRIEF DESCRIPTION OF THE FIGURES
[0047] FIG. 1 shows the effect of 2% propylene oxide at varying
temperatures between 15.degree. C. and 45.degree. C. on B. subtilis
spores over time.
[0048] FIG. 2 shows the effect of 4% propylene oxide at varying
temperatures between 15.degree. C. and 45.degree. C. on B. subtilis
spores over time.
DETAILED DESCRIPTION
[0049] Before describing the present invention in detail, it is to
be understood that this invention is not limited to particularly
exemplified methods of production, which may, of course, vary. It
is also to be understood that the terminology used herein is for
the purpose of describing particular embodiments of the invention
only, and is not intended to be limiting which will be limited only
by the appended claims.
[0050] All publications, patents and patent applications cited
herein, whether supra or infra, are hereby incorporated by
reference in their entirety. However, publications mentioned herein
are cited for the purpose of describing and disclosing the
protocols and reagents which are reported in the publications and
which might be used in connection with the invention.
[0051] Nothing herein is to be construed as an admission that the
invention is not entitled to antedate such disclosure by virtue of
prior invention.
[0052] Furthermore, the practice of the present invention employs,
unless otherwise indicated, conventional immunological techniques,
chemistry and pharmacology within the skill of the art. Such
techniques are well known to the skilled worker, and are explained
fully in the literature. See, e.g., Coligan, Dunn, Ploegh, Speicher
and Wingfield "Current protocols in Protein Science" (1999) Volume
I and II (John Wiley & Sons Inc.); and Bailey, J. E. and Ollis,
D. F., Biochemical Engineering Fundamentals, McGraw-Hill Book
Company, N.Y., 1986; Immunochemical Methods In Cell And Molecular
Biology (Mayer and Walker, eds., Academic Press, London, 1987);
Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and
C. C. Blackwell, eds., 1986).
[0053] It must be noted that as used herein and in the appended
claims, the singular forms "a," "an," and "the" include plural
reference unless the context clearly dictates otherwise.
[0054] Thus, for example, a reference to "a cross-linking agent"
includes a plurality of such agents, and a reference to "an
alcohol" is a reference to one or more alcohols, and so forth.
Unless defined otherwise, all technical and scientific terms used
herein have the same meanings as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any materials and methods similar or equivalent to those described
herein can be used to practice or test the present invention, the
preferred materials and methods are now described.
[0055] In one of the broadest aspects, the present invention
relates to a method for sterilizing a collagen-based
biomaterial.
[0056] As used herein, the term "biomaterial" refers to any
collagen containing material that potentially has a biological use.
The collagen might be any type of collagen from any source and
might be present alone or in combination with other materials.
Accordingly, the collagen might represent as little as 1% w/w of
the total weight of the biomaterial or as much as 100%.
[0057] The term "collagen" as used herein refers to the
extracellular family of fibrous proteins that are characterised by
their stiff, triple-stranded helical structure. Three collagen
polypeptide chains (".alpha.-chains") are wound around each other
to form this helical molecule. The term is also intended to
encompass the various types of collagen.
[0058] The major portion of the helical portion of collagen varies
little between mammalian species. Indeed, a number of collagen
types have high degrees of nucleotide and amino acid sequence
homologies. For example, the nucleotide sequence homology for
collagen alpha I type II is at least 88% when comparing humans,
equines and murine. Humans and equines have 93% sequence homology
at the nucleotide level, while mouse and equine have 89% sequence
homology. The nucleotide sequence homology for human and mouse is
88% (see, NCBI accession numbers U62528 (Equine), NM033150 (Human)
and NM031163 (mouse) http://www.ncbi.nlm.nih.gov). Other types of
collagen have similar levels of amino acid homology. For example,
the nucleotide sequence homology between porcine collagen alpha I
type I and ovine collagen alpha I type I is 90% (see, NCBI
accession numbers AF29287 (Ovine) and AF201723 (Porcine)
http://www.ncbi.nlm.nih.gov).
[0059] Given the level of common ancestry and biology for many of
the above animals, the high degree of amino acid and nucleotide
sequence homology for collagen across a number of species such as
cattle, sheep, mice and pigs, a person skilled in the art would
appreciate that the methods for producing the biomaterial as
disclosed herein are applicable for collagenous material isolated
from all mammalian animals.
[0060] Accordingly, in some embodiments, the biomaterial is
isolated or harvested from an animal of one of the mammalian orders
i.e. Artiodactyla, Lagomorpha, Rodentia, Perissodactyla, Carnivora
and Marsupialia. The animal is preferably an ovine, a bovine, a
caprine, an equine, a porcine, a marsupial or a human. While the
biomaterial is preferably isolated from the same animal species as
the recipient, it is envisaged that the biomaterial might be
isolated from a different species to the recipient.
[0061] Alternatively, in some embodiments, the biomaterial
comprises a cultured tissue, a reconstituted tissue or the
like.
[0062] The biomaterial might be any type of cellular tissue. For
example, the cellular tissue might be cardiovascular tissue, pelvic
floor tissue, heart tissue, heart valve, aortic roots, aortic wall,
aortic leaflets, pericardial tissue, connective tissue, the matrix
of soft or solid organs, dermal tissue, a vascular tissue, dura
mater, cartilage, pericardium, ligament, tendon blood vessels,
umbilical tissue, bone tissue, fasciae, and submucosal tissue or
skin as all of these comprises some collagen.
[0063] It will also be appreciated that the biomaterial might
further comprise synthetic analogs formed from synthetic polymers,
purified biological polymers, or both, including those generally
found in natural tissue matrices. Suitable synthetic polymers
include, for example, polyamides and polysulphones. Biological
polymers can be naturally occurring or produced in vitro by, for
example, fermentation and the like.
[0064] Purified biological polymers can be appropriately formed
into a substrate by techniques such as weaving, knitting, casting,
moulding, extrusion, cellular alignment, and magnetic alignment.
Suitable biological polymers include, without limitation, collagen,
elastin, silk, keratin, gelatin, polyamino acids, polysaccharides
(e.g. cellulose and starch), and copolymers of any of these. For
example, collagen and elastin polymers can be formed into a
synthetic implantable material by any of a variety of techniques,
such as weaving and moulding. Synthetic tissue analogs mimic a
natural tissue matrix. Alternatively, synthetic substrates can be
used to form a tissue analog, either alone or together with
naturally occurring substrates Non-limiting examples include,
polypropylene, polylactic acid, polyester, nylon, silicone and the
like.
[0065] Once the biomaterial has been acquired it is cross-linked
The cross-linking can utilize any of the well known procedures
including, but not limited to, those described in Eyre et al.,
1984, Annu. Rev. Biochem. 537, 717-748; Eyre, 1982, In: Symposium
on Heritable Disorders of Connective Tissue (Akeson et al. eds) pp.
43-58, Mosby, St. Louis, Mo.; Davison & Brennan, 1983, Connect.
Tissue Res. 11, 135-151; Robins, 1982, Methods Biochem. Analysis
28, 330-379; Reiser, 1991, Proc. Soc. Exp. Biol. Med. 196,
17-29.
[0066] A preferred method of cross-linking is disclosed in the
Applicants International Patent Application WO2006/066327
incorporated herein in its entirety by reference. Briefly, an
initial step in the preferred method of cross-linking the
collagen-based biomaterial of the present invention comprises
contacting the biomaterial with an alcohol-containing solution. As
used herein, the term "contacted," or "contacting" refers to the
active step of immersing the collagen-based biomaterial in a
solution or agent as described here, or as described infra,
subsequently contacting the biomaterial with a cross-linking agent,
an acidic solution or other matter for a sufficient period of time
to bring about a desired outcome. Methods for contacting the
biomaterial with, for example, the alcohol-containing solution are
well known in the art. For example, in general, the biomaterial can
be contacted by spraying, dipping or immersing the biomaterial in a
solution or agent.
[0067] The term "alcohol" as used herein refers to any alcohol
known in art which is capable of removing or reducing the amount of
triglycerides and at least partially esterifying the carboxyl
groups found on collagen. Preferably, the alcohol is a
water-soluble alcohol. More preferably, the alcohol is a
C.sub.1-C.sub.6 lower alcohol in a buffer-free solution. Even more
preferably, the alcohol is selected from the group consisting of
methanol, ethanol, cyclohexanol, isopropanol, propanol, butanol,
pentanol, isobutanol, sec-butanol and t-butanol.
[0068] Without wishing to be bound by any particular theory or
hypothesis the inventors consider that the alcohol-containing
solution assists in loosening the collagen triple helix and thereby
exposing hydrophobic sites (see, Karube & Nishida, 1979,
Biochim Biophys Acta., 23; 581(1): 106-13). They also consider that
the carboxyl and amine groups found in collagen are esterified in
the presence of the alcohol-containing solution such that they
become available for cross-linking in later steps. As such, a
preferred alcohol solution is one comprising at least about 50%
v/v, more preferably at least about 70% v/v and most preferably at
least about 80% v/v alcohol to buffer-free aqueous solution. In one
embodiment, the alcohol solution is 70% ethanol v/v in 0.9% saline
(containing 0.5 mM PMSF)
[0069] In some embodiments the alcohol-containing solution, as well
as other solutions and reagents used herein are "buffer-free" as it
is hypothesised that the cross-linking agents containing aldehyde
reacts with the buffer during fixation causing depolymerization of
the aldehyde.
[0070] The step of contacting the biomaterial to the
alcohol-containing solution may be carried out for any length of
time as long as it is sufficient to render the biomaterial
resistant to in vivo pathogenic calcification and that the majority
(i.e. a high percentage) of the carboxyl and amine groups found in
collagen are esterified. Preferably, the biomaterial remains in
contact with the alcohol-containing solution for sufficient time to
enable the alcohol to diffuse and permeate into the biomaterial.
More preferably, the biomaterial is exposed to the
alcohol-containing solution for at least 24 hours, even more
preferably at least 36 hours and most preferably, at least 48
hours.
[0071] Once the collagen-based biomaterial has been exposed to
alcohol it is removed. In some embodiments, the biomaterial is
rinsed after the exposure to alcohol in a rinsing solution
comprising a phosphate-free solution of 0.9% v/v saline. However,
any non-buffered physiologically acceptable solution may be used as
a rinsing solution. The purpose of the rinsing solution is mainly
to remove excess alcohol and as such is not critical.
[0072] After the collagen-based biomaterial has been exposed to
alcohol for greater than 24 hours it is then contacted with one or
more cross-linking agents, especially bifunctional cross-linking
agents. The term "bifunctional" as used herein refers to the two
functional aldehyde groups, present at both ends of the five carbon
chain. The cross-linking can be undertaken by any technique known
in the art, with any form of cross-linking agent as long as it is
capable of cross-linking collagen. Cross-linking agents, include
but are not limited to, acylating compounds, adipyl chloride,
aldehydes, alkyl and aryl halides, bisimidates, carbodiimides,
divinyl sulfone (DVS), formaldehyde, glutaraldehyde, glyoxal,
hexamethylene diisocyanate, hydroxychloride, hydroxyethyl
methacrylate divinyl sulfone (HEMA-DIS-HEMA), imidoesters,
isocyanates, light (e.g. blue light and UV light),
N-hydroxysuccinimide, N-substituted maleimides, pH, polyaldehyde,
diphenylphosphoryl azide (DPPA), polyepoxy compounds comprising
backbone of 17-25 carbons and 4-5 epoxy groups, polyepoxy ethers,
polyethylene glycol divinyl sulfone (VS-PEG-VS), polyglycerol
polyglycidyl ether and temperature and combinations thereof.
[0073] In some embodiments, the cross-linking agent is a chemical
cross-linking agent such as carbodiimide, polyepoxy ethers, divinyl
sulfone (DVS), genipin, glutaraldehyde, formaldehyde and
diphenylphosphoryl azide (DPPA).
[0074] It has also been demonstrated that polyepoxy compounds
comprising backbone of 17-25 carbons and 4-5 epoxy groups show a
high efficiency for the cross-linking collagen (see, for example,
US Pat. Applic. No. 20040059430 (Ser. No. 10/618,447). It has also
been shown that the toxicity of polyepoxy compounds is lower than
that of glutaraldehyde, and the antigenicity or immune-response
induction of tissues decreases in proportion to the reaction time,
in case of reacting with helical polypeptide molecules such as
collagen. Naturally, it shows relatively good biocompatibility
(see, for example, Lohre et al., (1992), Artif. Organs, 16:630-633;
Uematsu et al., (1998), Artif. Organs, 22:909-913). Consequently,
polyepoxy compounds as described are one preferred cross-linking
agent.
[0075] In some embodiments, the cross-linking agent comprises about
1% glutaraldehyde and the length of exposure is at least about 24
hours. It will be appreciated that the time length for exposure of
the biomaterial to the cross-linking agent depends on the agent
used, the concentration and the temperature. Typically, the length
of exposure is between 24 hours to 28 days. The determination of
the precise amount of exposure time for the biomaterial to the
cross-linking agent is well within the scope of a person skilled in
the art.
[0076] Again, without wishing to be bound by any particular theory
or hypothesis, the inventors consider that by exposing the
collagen-based biomaterial that has been exposed to alcohol to a
cross-linking agent, the esterified carboxyl groups and amine
groups on the collagen present in the biomaterial are
cross-linked.
[0077] While it will be appreciated by those skilled in the art
that the temperature at which each of the steps of the preferred
cross-linking method is carried out is not critical, it will be
understood that preferably, the temperature is between 2.degree. C.
and 40.degree. C., more preferably, between 4.degree. C. and
30.degree. C. and most preferably, between 5.degree. C. and
25.degree. C.
[0078] Once again, after the cross-linking step, the collagen-based
biomaterial is preferably rinsed in rinsing solution such as that
used after the alcohol exposure step (a). However, it will again be
appreciated that the rinsing step is merely a preferment.
[0079] Following the cross-linking step, or if utilised, the
rinsing step after the cross-linking step, the collagen-based
biomaterial may then be sterilized for use by the methods described
herein. Alternatively, the collagen-based biomaterial is contacted
with an acidic solution containing any acid capable of inactivating
and/or modifying the fixed and/or unfixed cross-linking agent
moieties present in the biomaterial after step (b) to remove or
reduce available calcium binding sites. Alternatively, or in
addition to, the acidic solution used in step (c) contains any acid
capable of further cross-linking the activated carboxyl groups with
the activated amine groups on the collagen to form amide bonds.
[0080] Preferably, the acidic solution comprises at least one
aminocarboxylic acid. The term "aminocarboxylic acid" as used
herein is any acid having at least one amino group and at least one
carboxylic acid substituent. Representative examples of
aminocarboxylic acids that are useful in the present invention
include, but are not limited to, L-glutamate, L-aspartate, L-lysine
L-arginine, L-histidine. The purpose of the acidic solution is
two-fold: firstly, the aminocarboxylic acid assists in the
inactivation and/or modification of the fixed and unfixed
cross-linking agent moieties, thereby reducing or alleviating any
adverse biological effects. Secondly, the aminocarboxylic acid
further cross-links the activated carboxyl groups with the
activated amine groups on the collagen to form amide bonds.
[0081] The concentration of the aminocarboxylic acid will depend
upon the actual acid used and other parameters such as total mass
of the biomaterial used and the like. In addition, a minimum wet
weight ratio of aminocarboxylic acid to biomaterial would be about
1:4. The most important aspect of the acidic solution is the pH.
The pH must be below pH7, preferably below pH6, more preferably
below pH5 and most preferably below about pH4.6.
[0082] In one embodiment, the acidic solution is 8 mg
aminocarboxylic acid per millilitre of de-ionised water, which is
phosphate-free and about pH4.
[0083] The cross-linked collagen-based biomaterial is exposed to
the aminocarboxylic acid for at least 6 hours, more preferably at
least 24 hours, even more preferably more than 48 hours. While the
incubation temperature is not critical it is preferably between
5.degree. C. and 55.degree. C., more preferably between 10.degree.
C. and 45.degree. C., most preferably about 45.degree. C.
[0084] In some embodiments, step (c) of the disclosed cross-linking
method is replaced by or supplemented with a method of inhibiting
the formation of metalloproteinase on elastin molecules present in
the biomaterial. Specifically, in tissue such as aortic tissue a
higher percentage of elastin is present than in other tissue. These
elastin molecules can provide sites for the formation of
metalloproteinase as such these sites need to be reduced, removed
or inactivated.
[0085] The cross-linked collagen-based biomaterial, before or after
the step of exposing the biomaterial to the acidic solution and/or
buffer-free solution containing a multi-valent cation, is again
preferably rinsed in rinsing solution. The cross-linked
collagen-based biomaterial is then sterilized.
[0086] The step of sterilizing the biomaterial comprises contacting
the cross-linked collagen-based biomaterial with a sterilization
solution comprising between 3% and 6% v/v propylene oxide and
incubating said biomaterial between 30.degree. C. and 55.degree. C.
for greater than 48 hours; with the proviso that the sterilization
solution does not include alcohol.
[0087] It will be appreciated that alcohol, especially ethanol
and/or isopropanol is not used in the sterilization solution of the
present invention.
[0088] It has been well established that at elevated temperatures
eg above 55.degree. C., collagen undergoes intracellular
degradation. Indeed, it has been shown that collagen within human
skin fibroblasts starts to undergo increased degradation at
temperatures above 41.degree. C. (Palotie, 1983, Coll Relat Res.
March; 3(2): 105-13). Thus, in sterilizing the cross-linked
collagen- based biomaterial of the present invention the
temperature of incubation is a critical factor. The temperature is
preferably not greater than 55.degree. C. as this increases the
chance that the collagen begins to degrade. However, as described
in Example 9 and elsewhere, it is important that the incubation
temperature is not less than 30.degree. C. as temperatures lower
than 30.degree. C. have reduced sterilization potential.
[0089] It will be appreciated by persons skilled in the art that
concentrations of propylene oxide below 3% would not provide
sufficient sterilization as defined herein. Concentrations of
propylene oxide above 6% are toxic and have an adverse effect on
the integrity of the biomaterial. In some embodiments, the
sterilization solution comprises between 3.8% and 4.5% propylene
oxide. In other embodiments, the sterilization solution comprises
about 4% propylene oxide. In some embodiments, the sterilization
solution consists essentially of between 3% and 6% propylene oxide,
more preferably the sterilization solution consists of between 3%
and 6% propylene oxide. In some embodiments, the sterilization
solution consists essentially of between 3.8% and 4.5% propylene
oxide, more preferably the sterilization solution consists of
between 3.8% and 4.5% propylene oxide. In some embodiments, the
sterilization solution consists essentially of about 4% propylene
oxide, more preferably the sterilization solution consists of about
4% propylene oxide.
[0090] The term "about" as used herein refers to a deviation in the
value following the term by 10% above or below. For example,
reference to about 4% propylene oxide includes ranges between 3.6%
and 4.4% i.e. 10% below or above the 4% value. This includes 3.7%,
3.8%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3% and 4.4% propylene oxide.
[0091] It is a requirement that the sterilization step is carried
out for greater than 48 hours; however, as described herein
propylene oxide can also be used as a storage media and as such the
sterilization step can be carried out for at least 2, 3, 4, 5, 6,
7, 8, 9, 10 days or more.
[0092] One major benefit of the methods described herein is that
the sterilization solution used herein i.e between 3% and 6% v/v
propylene oxide will not only sterilize collagen-containing tissue
without affecting the collagen fibrils, but as propylene oxide
converts after about 4 days being in contact with the biomaterial
to propylene glycol (which is not toxic), the sterilized
cross-linked collagen-based biomaterial can remain in the
sterilization solution well after the initial 48 hours. Indeed, it
is envisaged that the cross-linked collagen-based biomaterial will
be sterilized and stored and then shipped in the same container to
the end customer without the need for further handling.
[0093] The term "sterilization" as used herein means that the
cross-linked collagen-based biomaterial satisfies the requirements
under ISO 14160. ISO 14160 covers the sterilization of health care
products and pertains to liquid chemical sterilizing agents for
single-use medical devices utilizing animal tissues and their
derivatives. Briefly, ISO 14160 requires tissues to be inoculated
with B. subtilis spores and then treated to remove the
contamination. The requirements for ISO 14160 trials are described
in Example 6 supra.
[0094] In some embodiments, the sterilization solution is
buffer-free. In other embodiments the solution comprises de-ionized
water.
[0095] The cross-linked collagen-based biomaterial, after treatment
with the methods disclosed herein, has a high level of resistance
to calcification i.e. it is a "calcification-resistant
biomaterial". The term "calcification" as used herein refers to one
of the major pathological problems associated with traditionally
produced biomaterial comprising connective tissue proteins (i.e.,
collagen and elastin). It has previously been shown that these
materials can become calcified following implantation within the
body. Such calcification can result in undesirable stiffening or
degradation of the biomaterial. Two (2) types of calcification:
intrinsic and extrinsic are known to occur in fixed collagenous
biomaterial, although the exact mechanism(s) by which such
calcification occurs is unknown. Intrinsic calcification is
characterised by the precipitation of calcium and phosphate ions
within the fixed bioprosthetic tissue, including the collagen
matrix and remnant cells. Extrinsic calcification is characterised
by the precipitation of calcium and phosphate ions within adherent
thrombus, including adherent cells (e.g., platelets) to the
biomaterial and the development of calcium phosphate-containing
surface plaques on the biomaterial.
[0096] Consequently, the phrase "high level of resistance to
calcification" or "calcification-resistant" when applied to the
biomaterial of the present invention means that the biomaterial,
after in vivo implantation for at least 200 days, shows less than
50 preferably less than 20 .mu.g, and even more preferably less
than 10 .mu.g of calcium per mg of dried tissue after its
removal.
[0097] Preferably, the biomaterial of the present invention is also
resistant to enzymatic degradation. The term "resistant to
enzymatic degradation" as used herein refers to the ability of the
biomaterial of the present invention to withstand enzymatic
degradation to a comparable level with traditional fixed
tissue.
[0098] Once formed, the sterilized, cross-linked collagen-based
biomaterial of the present invention can then be used to treat a
number of conditions and/or disorders.
[0099] Generally, the terms "treating," "treatment" and the like
are used herein to mean affecting an individual or animal, their
tissue or cells to obtain a desired pharmacological and/or
physiological effect. The effect is especially therapeutic in terms
of a partial or complete cure of a condition and/or disorder.
"Treating" as used herein covers any treatment of a condition
and/or disorder in a vertebrate, a mammal, particularly a human,
and includes: (a) inhibiting the condition and/or disorder, i.e.,
arresting its development; or (b) relieving or ameliorating the
symptoms of the condition and/or disorder, i.e., cause regression
of the symptoms of the enzymatic degradation/condition and/or
disorder.
[0100] The terms "condition" and/or "disorder" are used herein
interchangeably and refers to abnormal conditions affecting
animals, including humans, which can be treated using the
biomaterial of the present invention. Accordingly, the treatment of
a wound, a lesion, tissue degeneration, a microbial infection, a
burn, an ulcer, dermal condition is included in the present
invention. Moreover, the replacement of heart valves, aortic roots,
aortic wall, aortic leaflets, pericardial tissue, connective
tissue, dura mater, dermal tissue, a vascular tissue, cartilage,
pericardium, ligaments, tendon blood vessels, umbilical tissue,
bone tissue, fasciae, and submucosal tissue are also
encompassed.
[0101] The calcification-resistant biomaterial of the present
invention may also be applied to any of a wide variety of
contacting surfaces of medical devices. Contacting surfaces
include, but are not limited to, surfaces that are intended to
contact blood, cells or other bodily fluids or tissues of an
animal, including specifically a human. Suitable contacting
surfaces include one or more surfaces of medical devices that are
intended to contact blood or other tissues. The medical devices
include aneurysm coils, artificial blood vessels, artificial
hearts, artificial valves, artificial kidneys, artificial tendons
and ligaments, blood bags, blood oxygenators, bone and
cardiovascular replacements, bone prostheses, bone waxes,
cardiovascular grafts, cartilage replacement devices, catheters,
contact lenses, containers for cell and tissue culture and
regeneration, embolization particles, filtration systems, grafts,
guide channels, in-dwelling catheters, laboratory instruments,
microbeads, nerve-growth guides, ophthalmic implants, orthopedic
implants, pacemaker leads, probes, prosthetics, shunts, stents,
supports for peptides, surgical instruments, sutures, syringes,
urinary tract replacements, wound coverings, wound dressings, wound
healing devices and other medical devices known in the art.
[0102] Other examples of medical devices that would benefit from
the application of the present invention will be readily apparent
to those skilled in the art of surgical and medical procedures and
are therefore contemplated by the instant invention. The contacting
surface may include a mesh, coil, wire, inflatable balloon, or any
other structure which is capable of being implanted at a target
location, including intravascular locations, intralumenal
locations, locations within solid tissue, and the like. The
implantable device can be intended for permanent or temporary
implantation. Such devices may be delivered by or incorporated into
intravascular and other medical catheters.
[0103] The process of coating the surfaces of such devices can be
performed by the plasma coating technique, as described in the
International patent application No. WO96/24392. By "comprising" is
meant including, but not limited to, whatever follows the word
comprising". Thus, use of the term "comprising" indicates that the
listed elements are required or mandatory, but that other elements
are optional and may or may not be present. By "consisting of" is
meant including, and limited to, whatever follows the phrase
"consisting of". Thus, the phrase "consisting of" indicates that
the listed elements are required or mandatory, and that no other
elements may be present. By "consisting essentially of" is meant
including any elements listed after the phrase, and limited to
other elements that do not interfere with or contribute to the
activity or action specified in the disclosure for the listed
elements. Thus, the phrase "consisting essentially of" indicates
that the listed elements are required or mandatory, but that other
elements are optional and may or may not be present depending upon
whether or not they affect the activity or action of the listed
elements.
[0104] The invention will now be further described by way of
reference only to the following non-limiting examples. It should be
understood, however, that the examples following are illustrative
only, and should not be taken in any way as a restriction on the
generality of the invention described above.
EXAMPLE 1
Basic Processing and Storage of Biomaterial
Harvesting of a Collagen-Derived Biomaterial
[0105] Porcine hearts from adult pigs were harvested at a local
abattoir and transported to the laboratory on ice packs within 2-4
hours of death. The hearts were washed twice in ice-cold 0.9% v/v
saline solution and carefully cleaned from adherent fat and loose
connective tissue. The aortic roots with the aortic valves were
dissected from the hearts and placed in ice-cold 0.9% v/v
saline/Phenyl-methyl-sulfonyl-fluoride (PMSF) and the valved aortic
roots washed for 20 minutes in the 0.9% v/v saline solution
containing PMSF. The valve leaflets were removed from the aortic
valve orifice and stored in ice-cold 0.9% v/v saline solution.
Cross-Linking (Fixation) of the Biomaterial
[0106] A 0.625% v/v glutaraldehyde solution containing 9.07 g/l
potassium di-hydrogen phosphate buffer in sterile, deionised water
was prepared. The pH of the glutaraldehyde solution was adjusted to
7.4 with sodium hydroxide. The aortic valve leaflets were
cross-linked in the glutaraldehyde solution at 1-5.degree. C. for a
minimum period of 5 days to crosslink proteins present in the
collagen of the tissues.
Rinsing the Cross-Linked Biomaterial
[0107] The aortic valve leaflets were removed from the
glutaraldehyde solution and rinsed in a sterile 0.9% v/v sodium
chloride for about 15 minutes. During the rinsing period, the
temperature of the rinsing solution was maintained at approximately
10.degree. C.
Final Sterilization and Storage of the Biomaterial
[0108] The porcine aortic valve leaflets were immersed in a 2.0%
v/v solution of glutaraldehyde containing 29.02 g/l potassium
di-hydrogen phosphate buffer in sterile, deionised water. The pH of
the aldehyde solution was adjusted to 7.4 with sodium hydroxide.
The process of sterilization was carried out at about 25.degree. C.
for 5 days. The sterilized tissues were divided into four groups
and stored in: (i) 0.625% v/v glutaraldehyde, (ii) 5.0% v/v
glutaraldehyde, (iii) 10% v/v glutaraldehyde; and (iv) 2% v/v
propylene oxide until further use.
EXAMPLE 2
Effect of Storage Solution on Calcification Profile of
Biomaterial
[0109] Experimental studies in small and large animal models were
conducted to assess the effectiveness of the above-described
sterilisation-storage process in mitigating calcification of
treated collagen containing biomaterials.
[0110] In the first animal study, porcine aortic valve leaflets
sterilised and stored according to the methods described in Example
1 were used for assessment in a small animal model.
[0111] Sterilised and stored porcine aortic valve leaflets of all
four groups were rinsed in 0.9% v/v saline for 5 minutes. The
rinsed tissues were surgically implanted in subcutaneous pockets
(one sample of each group per rat), created in the central
abdominal wall area of growing (6 weeks old) male Wistar rats.
These tissues were removed after 60 days, host tissue removed and
samples dried in a Biotherm.TM. incubator (Marcus Medical, JHB,
RSA) at 90.degree. C. for 48 h. The dried samples were weighed, and
the calcium content extracted in 5.0 ml 6 N ultrapure hydrochloric
acid (Merck, JHB, RSA) at 75.degree. C. for 24 h. The extractable
calcium content was then measured using an atomic absorption
spectrophotometer (Varian AA1275) and expressed as .mu.g calcium
per mg tissue (dry weight). These data are summarised in Table 1.
Results (.mu.g Calcium per mg dried tissue) are summarised in Table
1.
TABLE-US-00001 TABLE 1 Storage solution Mean .+-.standard error
Glutaraldehyde (0.625%) 70.146 .mu.g Ca/mg Tissue .+-.7.037
Glutaraldehyde (5.0%) 88.439 .mu.g Ca/mg Tissue .+-.4.470
Glutaraldehyde (10.0%) 66.870 .mu.g Ca/mg Tissue .+-.13.235
Propylene Oxide (2.0%) 25.311 .mu.g Ca/mg Tissue .+-.5.292
EXAMPLE 3
Effect of Sterilization & Storage Solution on Calcification
Profile of Biomaterial
Harvesting of a Collagen-Ferived Biomaterial
[0112] In the second animal study, porcine aortic valve leaflets
were harvested and isolated according to the method described in
Example I. Isolated porcine aortic valve leaflets were divided into
three groups. Group I received a typical cross-linking treatment
(control); Group II received a proprietary method of cross-linking
(see WO2006/066327 incorporated herein by reference); and Group III
received the same cross-linking treatment as Group II, but this was
followed by the incubating the cross-linked biomaterial with a
sterilization solution comprising about 4% v/v propylene oxide and
incubating the biomaterial between 30.degree. C. and 55.degree. C.
for greater than 48 hours.
Cross-Linking (Fixation) of the Aortic Valve Leaflets
[0113] In group I, porcine aortic valve leaflets were cross-linked
in a 0.625% glutaraldehyde solution containing 9.07 g/l potassium
di-hydrogen phosphate buffer in sterile, deionised water was
prepared. The pH of the glutaraldehyde solution was adjusted to 7.4
with sodium hydroxide. The aortic valve leaflets were cross-linked
in the glutaraldehyde solution at 1-5.degree. C. for a minimum
period of 5 days to crosslink proteins present in the collagen of
the tissues.
[0114] In group II and III, a water-soluble alcohol-containing
solution of 60-80% v/v by volume alcohol ethanol was prepared. The
porcine aortic valve leaflets were immersed into the alcohol
solution after overnight storage at 4.degree. C. The valved aortic
roots were immersed in the same alcohol solution immediately after
the final wash in ice-cold 0.9% v/v saline (containing 0.5 mM
PMSF). The porcine aortic valve leaflets were kept in the alcohol
solution at about 5.degree. C. for a minimum of 24 hours.
[0115] The porcine aortic valve leaflets were removed from the
alcohol solution and rinsed for about 10 minutes with 0.9% v/v
saline. During the rinsing period, the temperature of the rinsing
solution was maintained at approximately 10.degree. C.
[0116] The aortic valve leaflets were immersed in a 0.625% v/v
solution of glutaraldehyde containing 9.07 g/l potassium
di-hydrogen phosphate buffer in sterile, deionised water. The pH of
the glutaraldehyde solution was adjusted to 7.4 with sodium
hydroxide. The pericardium and the valved aortic roots were fixed
in the glutaraldehyde solution at 1-5.degree. C. for a minimum
period of 24 hours to crosslink proteins present in the collagen of
the tissues.
[0117] The porcine valve leaflets were removed from the
glutaraldehyde solution and rinsed in a sterile 0.9% v/v sodium
chloride for about 15 minutes. During the rinsing period, the
temperature of the rinsing solution was maintained at approximately
10.degree. C.
[0118] The porcine aortic valve leaflets were then immersed in a
buffer-free solution containing 8 mg dicarboxylic acid per 1 ml
de-ionised water volume. The pH of the solution was adjusted to a
pH of 4.5 with a volume of diluted hydrochloric acid. The
pericardium and the valved aortic roots were immersed in the
solution at a temperature of about 45.degree. C. for about 48
hours.
Final Sterilization and Storage of the Biomaterial
[0119] The porcine aortic valve leaflets were then sterilized and
stored either by: [0120] (i) immersing the tissue in a 0.25% v/v
solution of glutaraldehyde containing 9.07 g/l potassium
di-hydrogen phosphate buffer in sterile, deionised water. The pH of
the aldehyde solution was adjusted to 7.4 with sodium hydroxide.
The process of sterilization was carried out at a temperature about
45.degree. C. for about 120 minutes (Treatment A); or [0121] (ii)
the porcine aortic valve leaflets were sterilized in an aqueous
solution comprising of 4% v/v propylene Oxide by weight combined
with 20% v/v ethyl alcohol at 37.degree. C. for about 24 hours and
stored in a 4% v/v propylene oxide solution Treatment B--present
invention).
[0122] Sterilized and stored porcine aortic valve leaflets of all
three groups were rinsed in 0.9% v/v saline for 5 minutes. The
rinsed tissues were surgically implanted in subcutaneous pockets
(one sample of each group per rat), created in the central
abdominal wall area of growing (6 weeks old) male Wistar rats.
These tissues were removed after 60 days, host tissue removed and
samples dried in a Biotherm.TM. incubator (Selby Scientific, Perth,
Wash.) at 90.degree. C. for 48 h. The dried samples were weighed,
and the calcium content extracted in 5.0 ml 6 N ultrapure
hydrochloric acid (Merck, Sydney, Australia) at 75.degree. C. for
24 h. The extractable calcium content was then measured using an
atomic absorption spectrophotometer (Varian AA1275) and expressed
as .mu.g calcium per mg tissue (dry weight). Results (.mu.g Calcium
per mg dried tissue) are summarised in Table 2.
TABLE-US-00002 TABLE 2 Storage solution Mean .+-.standard error
Glutaraldehyde (0.625%) 174.525 .mu.g Ca/mg Tissue .+-.6.884
Treatment A 3.300 .mu.g Ca/mg Tissue .+-.0.289 0.25% Glutaraldehyde
Treatment B 1.325 .mu.g Ca/mg Tissue .+-.0.317 4% propylene
Oxide
EXAMPLE 4
Effect of Treatment B on Calcification Profile of Bovine
Pericardium
[0123] In third animal study, the calcification potential of bovine
pericardium prepared, cross-linked and stored according to the
tissues in Example 3 (0.625% buffered glutaraldehyde, Treatment
A+0.2% glutaraldehyde and Treatment B 4% v/v propylene oxide) was
compared with the calcification potential of commercial bovine
pericardium (Hancock pericardium) stored in a 0.2% glutaraldehyde
solution.
[0124] Representative samples of each group were trimmed to
1.times.1 cm size and rinsed in 0.9% v/v saline for 5 minutes.
These samples were surgically implanted in subcutaneous pockets,
created in the central dorsal wall area of growing (6 weeks old)
male Wistar rats. These tissues were removed after 60 days, host
tissue removed and the calcium content determined by atomic
absorption spectrophotometry. Results (.mu.g Calcium per mg dried
tissue) are summarised in Table 3.
TABLE-US-00003 TABLE 3 Storage solution Mean .+-.standard error
Glutaraldehyde (0.625%) 136.025 .mu.g Ca/mg Tissue .+-.11.385 ADAPT
+ 4.100 .mu.g Ca/mg Tissue .+-.0.204 0.25% Glutaraldehyde ADAPT +
1.100 .mu.g Ca/mg Tissue .+-.0.147 4% v/v Propylene Oxide Hancock
Pericardium 6.375 .mu.g Ca/mg Tissue .+-.1.993 (in 0.2%
Glutaraldehyde)
EXAMPLE 5
Effect of Treatment B on Calcification Profile of Porcine Aortic
Valve Tissue (Valve Leaflets & Aortic Wall) in a Large Animal
Model
[0125] In the fourth animal study, the calcification potential of
porcine aortic valve tissue (valve leaflets and aortic wall)
prepared, cross-linked in 0.625% buffered glutaraldehyde and stored
in (i) 0.625% glutaraldehyde, (ii) treated with Treatment A (0.625%
glutaraldehyde) and (iii) treated with Treatment B (4% propylene
oxide).
[0126] Representative samples of each group were trimmed to an oval
shaped size of approximately 1.2.times.1 cm and rinsed in 0.9%
saline for 5 minutes. These samples were surgically implanted in
the jugular vein of juvenile sheep (body weight 22-25 kg). These
tissues were removed after 150 days, host tissue removed and the
calcium content determined by atomic absorption spectrophotometry.
Results (.mu.g Calcium per mg dried tissue) are summarised in Table
4-A (Valve leaflets) and Table 4-B (Aortic wall).
TABLE-US-00004 TABLE 4-A (Valve leaflets) Storage solution Mean
.+-.standard error 0.625% Glutaraldehyde 211.100 .mu.g Ca/mg Tissue
.+-.3.134 Propylene Oxide 93.167 .mu.g Ca/mg Tissue .+-.23.764
Treatment B 12.775 .mu.g Ca/mg Tissue .+-.12.442 (4% propylene
oxide)
TABLE-US-00005 TABLE 4-B (Aortic wall) Storage solution Mean
.+-.standard error 0.625% Glutaraldehyde 59.444 .mu.g Ca/mg Tissue
.+-.12.263 2% Propylene Oxide 28.633 .mu.g Ca/mg Tissue .+-.8.370
Treatment B 18.287 .mu.g Ca/mg Tissue .+-.7.305 (4% propylene
oxide)
EXAMPLE 6
Validation: Sterilisation of Commercial Heart Valve Inoculated with
Bacillus Subtilis Spores
[0127] This validation was performed to test the feasibility of
sterilising commercial heart valve tissue with 4% propylene oxide
after 48 hours at 45.degree. C. The purpose of this feasibility
study was to investigate if 3.8% propylene oxide (as a "worst-case"
concentration level) is capable of sterilising commercial heart
valves X tissue under "worst-case" conditions (contamination with
Bacillus subtilis spores) prescribed by FDA regulations. The test
conditions were: [0128] The valves were removed from the 0.5%
Glutaraldehyde and rinsed in a total of 1000 mls of sterile
distilled water for a total of 6 mins. [0129] The valve holder and
the valve were then aseptically separated and then dried for
approximately 30 mins or until visibly dry. [0130] The valve holder
and the valve of each device were then inoculated with a total of
20 .mu.l of a suspension of Bacillus subtilis spores obtained from
STEMS Corporation, USA. The suspension contained
1.25.times.10.sup.6 spores. [0131] The valves were then allowed to
dry for approximately 1 hour at room temperature. [0132] The
devices were then reassembled as per receipt and placed into a
sterile jar. [0133] To ten devices, 160 mls of freshly prepared
3.8% propylene oxide was added. [0134] To the final device, 160 mls
of Soybean-Casein Digest Medium (SCDM) was added. This was the
positive control to assess the viability of the spore suspension.
The positive control was incubated at 32.degree. C. for 48 hours.
[0135] The ten test valves were then incubated at 42.degree. C. for
44 hours. [0136] Following incubation, a sterility test was
performed on each valve. [0137] The valves were separated and each
component transferred to an empty sterile jar, to which SCDM was
added. [0138] The jars were then incubated at 32.degree. C. for 14
days. [0139] The jars were examined daily for signs of
turbidity.
TABLE-US-00006 [0139] TABLE 5 Test Results SCDM No growth detected
after 14 days incubation at 32.degree. C. Stasis Test: Performed at
expiration of test period. SCDM showed visible growth of C.
albicans within 48 hours. Positive Control Growth detected after 24
hours. Growth identified as B. subtilis.
EXAMPLE 7
Effect of Treatment B on Calcification Profile of Commercial Heart
Valve Tissue (Bovine Pericardial Tissue) in a Small Animal
Model
[0140] Table 6 shows the results of a fifth animal study in which
the calcification potential of bovine pericardium cross-linked and
sterilised in 0.625% v/v glutaraldehyde (which served as a
reference control--marked A) was compared with commercial heart
valve tissue (bovine pericardium, cross-linked and stored according
to a commercial proprietary protocol which is 0.625% v/v buffered
glutaraldehyde cross-linking+formaldehyde storage--marked B) and
the same commercial heart valve tissue sterilised at 45.degree. C.
for 48 hours in 4% v/v propylene oxide and stored in 4% v/v
propylene oxide solution--marked C.
[0141] Representative samples of each group were trimmed to
1.times.1 cm size and rinsed in 0.9% v/v saline for 5 minutes.
These samples were surgically implanted in subcutaneous pockets,
created in the central dorsal wall area of growing (6 weeks old)
male Wistar rats. These tissues were removed after 8, 16 and 24
weeks, host tissue removed and the calcium content determined by
atomic absorption spectrophotometry. Results (.mu.g Calcium per mg
dried tissue) are summarised in Table 6.
TABLE-US-00007 TABLE 6 Storage solutions A B C 8 Weeks 85 .mu.g
Ca/mg Tissue .+-. 12 12 .mu.g Ca/mg Tissue .+-. 11 0.751 .mu.g
Ca/mg Tissue .+-. 0.2 16 Weeks 94 .mu.g Ca/mg Tissue .+-. 12 10
.mu.g Ca/mg Tissue .+-. 8 0.74 .mu.g Ca/mg Tissue .+-. 0.2 24 Weeks
134 .mu.g Ca/mg Tissue .+-. 12 8 .mu.g Ca/mg Tissue .+-. 6 3.56
.mu.g Ca/mg Tissue .+-. 3
EXAMPLE 8
Effect of Treatment B on Calcification Profile of Commercial Heart
Valve Tissue (Bovine Pericardial) Tissue in a Rapid In Vitro
Calcification Model
[0142] In a further experimental assessment, the calcification
potential of commercial valve tissue (control tissue) was compared
with commercial heart valve tissue sterilised at 45.degree. C. for
48 hours in 4% propylene oxide and stored in 4% propylene oxide
solution (treated tissue) in a rapid in vitro calcification
model.
[0143] Stented commercial heart valves (control and treated) were
mounted in a Rowan Ash Fatigue tester and exposed to a
physiological solution (with a high calcium/phosphate content)
during accelerated flow (400 test cycles per minute) up to 50
million cycles.
[0144] After 50 million test cycles, heart valves were removed and
a represented tissue sample taken for histology. The remaining
tissue of each of the three valve leaflets in each valve were
removed and the calcium content determined by atomic absorption
spectrophotometry. Results (.mu.g Calcium per mg dried tissue) are
summarised in Table 7.
TABLE-US-00008 TABLE 7 Valve tissue Mean .+-.standard error
Commercial valve 49.71 .mu.g Ca/mg tissue .+-.2.112 Commercial
valve + 32.34 .mu.g Ca/mg Tissue .+-.1.336 4% Propylene Oxide
EXAMPLE 9
Effect of Sterilization and Storage Methodology Tissue Inoculated
with Bacillus subtilis Spores
[0145] FIGS. 1 and 2 show the effect of 2% v/v and 4% v/v propylene
oxide (respectively) at varying temperatures between 15.degree. C.
and 45.degree. C. on B. subtilis spores over time. The experiment
conditions used are described in Example 6. Essentially, it can be
seen the neither sterilization solutions (2% or 4%) has little
sterilization effect before 48 hours. It can also be seen from FIG.
2 that within 48 hours the effect of increasing temperature has a
profound effect on sterilization. For example, at a temperature of
40.degree. C. and above there was sterilization after 24 hours and
that by 48 hours there was sterilization even at temperatures of
25.degree. C. and above. FIG. 1 shows that in order to obtain
sterilization with 2% v/v propylene oxide the tissue needs to be
incubated for at least 6 days at temperatures above 35.degree. C.
Even incubation for 10 days at 15 to 20.degree. C. has no material
effect on sterilization with 2% v/v propylene oxide.
[0146] Thus, it can be seen from FIGS. 1 and 2 that optimal
sterilization is obtained by incubating the tissue with a 4% v/v
propylene oxide solution and incubating the tissue at about
45.degree. C. for greater than 48 hours.
* * * * *
References